Advancing biodiversity assessments with environmental DNA: Long‐read technologies help reveal the drivers of Amazonian fungal diversity

Fungi are a key component of tropical biodiversity. However, due to their inconspicuous and largely subterranean nature, they are usually neglected in biodiversity inventories. The goal of this study was to identify the key determinants of fungal richness, community composition, and turnover in tropical rainforests. We tested specifically for the effect of soil properties, habitat, and locality in Amazonia. For these analyses, we used high‐throughput sequencing data of short and long reads of fungal DNA present in soil and organic litter samples, combining existing and novel genomic data. Habitat type (phytophysiognomy) emerges as the strongest factor explaining fungal community composition. Naturally open areas—campinas—are the richest habitat overall. Soil properties have different effects depending on the soil layer (litter or mineral soil) and the choice of genetic marker. We suggest that campinas could be a neglected hotspot of fungal diversity. An underlying cause for their rich diversity may be the overall low soil fertility, which increases the reliance on biotic interactions essential for nutrient absorption in these environments, notably ectomycorrhizal fungi–plant associations. Our results highlight the advantages of using both short and long DNA reads produced through high‐throughput sequencing to characterize fungal diversity. While short reads can suffice for diversity and community comparison, long reads add taxonomic precision and have the potential to reveal population diversity.     

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle-scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long-range polymerase chain reaction and using next-generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel-dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel-dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.     

From barcodes to genomes: extending the concept of DNA barcoding

DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach.     

Applications of sequencing technology in clinical microbial infection

Infectious diseases are a type of disease caused by pathogenic microorganisms. Although the discovery of antibiotics changed the treatment of infectious diseases and reduced the mortality of bacterial infections, resistant bacterial strains have emerged. Anti‐infective therapy based on aetiological evidence is the gold standard for clinical treatment, but the time lag and low positive culture rate of traditional methods of pathogen diagnosis leads to relative difficulty in obtaining the evidence of pathogens. Compared with traditional methods of pathogenic diagnosis, next‐generation and third‐generation sequencing technologies have many advantages in the detection of pathogenic microorganisms. In this review, we mainly introduce recent progress in research on pathogenic diagnostic technology and the applications of sequencing technology in the diagnosis of pathogenic microorganisms. This review provides new insights into the application of sequencing technology in the clinical diagnosis of microorganisms.     

A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well‐equipped molecular laboratory and is time‐consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION? and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION? reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch‐free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION? runs that represent three different stages of MinION? development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION? barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION? barcodes have an accuracy of 99.3%–100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be 相似文献   

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.     

Amplicon_sorter: A tool for reference‐free amplicon sorting based on sequence similarity and for building consensus sequences

Oxford Nanopore Technologies (ONT) is a third‐generation sequencing technology that is gaining popularity in ecological research for its portable and low‐cost sequencing possibilities. Although the technology excels at long‐read sequencing, it can also be applied to sequence amplicons. The downside of ONT is the low quality of the raw reads. Hence, generating a high‐quality consensus sequence is still a challenge. We present Amplicon_sorter, a tool for reference‐free sorting of ONT sequenced amplicons based on their similarity in sequence and length and for building solid consensus sequences.     

Assembly and analysis of a qingke reference genome demonstrate its close genetic relation to modern cultivated barley

Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third‐generation sequencer (PacBio), we de novo assembled a 4.84‐Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59‐Gb sequence to seven chromosomes. Of the 46,787 annotated ‘high‐confidence’ genes, 31 564 were validated by RNA‐sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by nonredundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low‐diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.     

Arbuscular mycorrhizal fungi: a specialised niche for rhizospheric and endocellular bacteria

Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria. The aim of this paper is to review briefly the interactions shown by these symbiotic fungi with two bacterial groups: (i) the plant-growth promoting rhizobacteria (PGPRs) which are usually associated with fungal surfaces in the rhizosphere, and (ii) a group of endocellular bacteria, previously identified as being related to Burkholderia on the basis of their ribosomal sequence strains. The endobacteria have been found in the cytoplasm of some isolates of AM fungi belonging to Gigasporaceae and offer a rare example of bacteria living in symbiosis with fungi. This revised version was published online in August 2006 with corrections to the Cover Date.     

A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data

Deep sequencing of the 16S rRNA gene provides a comprehensive view of bacterial communities in a particular environment and has expanded our ability to study the impact of the microflora on human health and disease. Current analysis methods rely on comparisons of the sequences generated with an expanding but limited set of annotated 16S rRNA sequences or phylogenic clustering of sequences based on arbitrary similarity cutoffs. We describe a novel approach to characterize bacterial composition using deep sequencing of 16S rRNA gene. Our method defines operational taxonomic units based on phylogenetic tree reconstruction and dynamic clustering of sequences using solely sequencing data. These OTUs can be used to identify differences in bacteria abundance between environments. This approach can perform better than previous phylogenetic methods and will significantly improve our understanding of the microfloral role on human diseases by providing a comprehensive analysis of the microbial composition from various bacterial communities.     

Beyond DNA barcoding: The unrealized potential of genome skim data in sample identification

Genetic tools are increasingly used to identify and discriminate between species. One key transition in this process was the recognition of the potential of the ca 658bp fragment of the organelle cytochrome c oxidase I (COI) as a barcode region, which revolutionized animal bioidentification and lead, among others, to the instigation of the Barcode of Life Database (BOLD), containing currently barcodes from >7.9 million specimens. Following this discovery, suggestions for other organellar regions and markers, and the primers with which to amplify them, have been continuously proposed. Most recently, the field has taken the leap from PCR‐based generation of DNA references into shotgun sequencing‐based “genome skimming” alternatives, with the ultimate goal of assembling organellar reference genomes. Unfortunately, in genome skimming approaches, much of the nuclear genome (as much as 99% of the sequence data) is discarded, which is not only wasteful, but can also limit the power of discrimination at, or below, the species level. Here, we advocate that the full shotgun sequence data can be used to assign an identity (that we term for convenience its “DNA‐mark”) for both voucher and query samples, without requiring any computationally intensive pretreatment (e.g. assembly) of reads. We argue that if reference databases are populated with such “DNA‐marks,” it will enable future DNA‐based taxonomic identification to complement, or even replace PCR of barcodes with genome skimming, and we discuss how such methodology ultimately could enable identification to population, or even individual, level.     

LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias

We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.     

Parallel barcoding of antibodies for DNA‐assisted proteomics

DNA‐assisted proteomics technologies enable ultra‐sensitive measurements in multiplex format using DNA‐barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio‐conjugation protocols. Here, we introduce a magnetic bead‐assisted DNA‐barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand‐fold. The success of DNA‐barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno‐PCR assays. Specific DNA‐barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read‐out on a massively parallel sequencing platform in a procedure denoted Immuno‐Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.     

A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats

Indigenous communities of soil‐resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under‐exploitation as soil health indicators, and a user‐friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress‐sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full‐length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA‐based systematics, Mononchida families (indicated here as M1–M5) are relatively well‐supported and, consequently, family‐specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (≈ 1000 bp from the 5′‐end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1–D9, PP1–PP3) that shows only distant similarity with ‘classical’ Dorylaimid systematics. Most subclades were trophically homogeneous, and — in most cases — specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real‐time polymerase chain reaction). Our results constitute proof‐of‐principle for the concept of DNA sequence signatures‐based monitoring of stress sensitive nematode families in environmental samples.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

DNA degradation in fish: Practical solutions and guidelines to improve DNA preservation for genomic research

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.     

Impacts of long‐term elevated atmospheric CO2 concentrations on communities of arbuscular mycorrhizal fungi

The ecological impacts of long‐term elevated atmospheric CO₂ (eCO₂) levels on soil microbiota remain largely unknown. This is particularly true for the arbuscular mycorrhizal (AM) fungi, which form mutualistic associations with over two‐thirds of terrestrial plant species and are entirely dependent on their plant hosts for carbon. Here, we use high‐resolution amplicon sequencing (Illumina, HiSeq) to quantify the response of AM fungal communities to the longest running (>15 years) free‐air carbon dioxide enrichment (FACE) experiment in the Northern Hemisphere (GiFACE); providing the first evaluation of these responses from old‐growth (>100 years) semi‐natural grasslands subjected to a 20% increase in atmospheric CO₂. eCO₂ significantly increased AM fungal richness but had a less‐pronounced impact on the composition of their communities. However, while broader changes in community composition were not observed, more subtle responses of specific AM fungal taxa were with populations both increasing and decreasing in abundance in response to eCO₂. Most population‐level responses to eCO₂ were not consistent through time, with a significant interaction between sampling time and eCO₂ treatment being observed. This suggests that the temporal dynamics of AM fungal populations may be disturbed by anthropogenic stressors. As AM fungi are functionally differentiated, with different taxa providing different benefits to host plants, changes in population densities in response to eCO₂ may significantly impact terrestrial plant communities and their productivity. Thus, predictions regarding future terrestrial ecosystems must consider changes both aboveground and belowground, but avoid relying on broad‐scale community‐level responses of soil microbes observed on single occasions.